The Role of Human Osteopontin Isoforms in Vascular Smooth Muscle Cell Migration and Cell Signaling Open Access

Harirforoosh, Sarah (2016)

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Obstructive arterial disease is characteristic of several cardiovascular disease (CVD) pathologies and ultimately leads to tissue ischemia. The endogenous response to ischemia is to increase collateral formation to restore blood flow and re-establish oxygen delivery to damaged tissue. Collateral formation involves the proliferation and migration of multiple cell types, including vascular smooth muscle cells (VSMCs). Osteopontin (OPN) is a secreted inflammatory protein that is significantly upregulated in response to ischemia. In humans, OPN is alternatively spliced to produce three isoforms: OPNa, OPNb, and OPNc. Several studies show that OPN isoform functions vary based on the tumor subtype, and data from the Lyle lab demonstrate that OPN isoforms diverge in their impacts on functional collateral formation in vivo. The migration of VSMCs requires several processes and steps to occur in concert. For example, during the formation of lamellipodia, focal adhesion kinase (FAK) must be activated to aid in the maturation of focal contacts into focal adhesions. VSMC migration requires lamellipodia and filipodia formation and persistence, cell polarization, and appropriate focal adhesion dynamics. Using several assays to assess cell migration, including a modified Boyden chamber assay, live cell imaging, and scratch wound assay experiments, we establish that OPN is required for proper VSMC migration. OPN-/- VSMCs exhibit impaired migration in response to PDGF, display deficits in lamellipodia formation and persistence, and close a smaller percentage of a wound compared to WT VSMCs. By using live cell imaging with the incorporation of a scratch wound assay, we were able to further determine that human osteopontin isoforms a and c rescue impaired OPN-/- VSMC migration. This project investigates the specific mechanisms by which OPN regulates VSMC migration. We establish that human OPN isoforms differentially rescue VSMC migration and identify potential mechanisms that may explain these differences. By understanding the mechanisms by which human OPN isoforms modulate cell migration, we may be able to utilize these isoforms as novel therapeutic targets for cardiovascular disease pathologies.

Table of Contents

Introduction 1

1.1 Cardiovascular Disease 2

1.2 Ischemia and its Physiological Response 2

1.3 Role of VSMCs in Collateral Formation, Atherosclerosis, and Arteriogenesis 3

1.4 Proper Collateral Formation Requires Osteopontin 3

1.4.1 Osteopontin 4

1.5 Human OPN Isoforms 5

1.5.1 Expression and Function of Human OPN Isoforms in Pathological Processes 7

1.6 Processes of Cell Migration 7

Methods 10

2.1 Cell Culture 11

2.2 Antibodies 11

2.3 Boyden Chamber Assay 11

2.4 Scratch Wound Assay 12

2.5 Live Cell Imaging with and without Scratch Wound Assay 13

2.6 Recombinant hOPN Protein 14

2.7 Cell Stimulation for Dose Response 14

2.8 Immunoblotting 16

2.9 Statistical Analysis 16

Experimental Results 17

3.1 OPN-/- VSMCs Exhibit Impaired Migration 18

3.2 OPN-/- VSMCs May Exhibit Impaired Lamellipodial Protrusion and Persistence 20

3.3 OPN-/- VSMCs Migration is Rescued by Human Osteopontin Isoforms a and c 25

3.4 Effects of Human OPN isoforms on FAK Phosphorylation 27

Discussion 30

References 36

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